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The conformation and stabilities of the CL fragment isolated from a type lambda Bence Jones protein and the fragment in which the intrachain disulfide bond had been reduced were studied by measuring CD, fluorescence, and ultraviolet absorption. The results indicated that no great conformational change occurs on reduction of the disulfide, unless the SH groups are alkylated. Intact CL was more resistant than reduced CL to guanidine hydrochloride. The denaturation curves were analyzed using an equation based on the binding of guanidine hydrochloride and the free energy changes of denaturation in the absence of the denaturant were estimated as about 6 kcal.mol-1 for intact CL and about 1.8 kcal.mol-1 for reduced CL. The difference in stability between intact CL and reduced CL was explained to a great extent in terms of the entropy change associated with reduction of the intrachain disulfide bond of the fragment in the denatured state.

SynopsisThe Raman spectra of Bence-Jones proteins (BJP) were measured for their native and denatured states. All of the native BJPs investigated gave amide I at 1670-1675 cm-l and amide I11 at 1242-1246 cm-'. Although the amide I was shifted to 1667 cm-l upon the LiBr, acid, and thermal denaturation, as expected, the amide 111 frequency was unaltered, indicating that the antiparallel p-and disordered structures of BJP provide amide I11 at almost the same frequencies. The intensity of the 880-cm-l line of native BJF' was relatively intense compared with that of amino acid mixed solution in which the mole ratios of Trp, Phe, and Tyr were adjusted to reproduce the corresponding ratios of BJP. However, the intensity was evidently reduced upon LiBr, acid, and thermal denaturation, approaching that of the amino acid mixture. Thus, the intensity of the 880-cm-' line is proposed as a practical probe for the environment of Trp residues. The pH dependence of the intensity of the 880-cm-' line suggests that one of two buried Trp residues is exposed between pH 4 and 3.2 and the other between pH 3.2 and 1.4. The variable fragment (VL) of BJF' (Tod) exhibited a S-S stretching Raman line at 525 cm-l. Provided that the crystallographic data of the VL of BJP is applicable to VL of BJP (Tod), the 525 cm-I of the S-S stretching frequency should be assigned to a TGG conformation of -C+SfSfC-linkage, but not to the AGT or AGG conformation. This supports Sugeta's model rather than Scheraga's model.

By limited proteolysis of a type kappa immunoglobulin light chain (Oku) with clostripain, both the VL and CL fragments were obtained with a high yield. The unfolding and refolding by guanidine hydrochloride of light chain Oku and its VL and CL fragments were studied at pH 7.5 and 25 degrees C with circular dichroism and tryptophyl fluorescence. The concentration of guanidine hydrochloride at the midpoint of the unfolding curve was 1.2 M for the VL fragment and 0.9 M for the CL fragment. As in the case of the CL fragment of light chain Nag (type lambda) [Goto, Y., & Hamaguchi, K. (1982) J. Mol. Biol. 156, 891-910], the unfolding and refolding kinetics of the CL fragment could be explained in principle on the basis of the three-species mechanism U1 in equilibrium U2 in equilibrium N, where N is native protein and U1 and U2 are the slow-folding and fast-folding species, respectively, of unfolded protein. The unfolding and refolding kinetics of the VL(Oku) fragment were described by a single exponential term. Double-jump experiments, however, showed that two forms of the unfolding molecule exist. The equilibrium and kinetics of unfolding of light chain Oku were explained by the sum of those of the VL and CL fragments. On the other hand, the refolding kinetics of light chain Oku were greatly different from the sum of those of the VL and CL fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

The pH difference absorption spectra of human lysozyme [EC 3.2.1.17] were measured. The difference spectra in the acidic region had a peak at 300 nm, as observed for hen and turkey lysozymes. The pH dependence curve of the extinction difference at 300 nm was well interpreted in terms of the pK values of the catalytic groups (3.4 for Asp 52 and 6.8 for Glu 35 at 0.1 ionic strength and 25 degrees C) determined from the pH dependence of the circular dichroism at 303.5 nm (Kuramitsu et al. (1974) J. Biochem. 76, 671--683) and the fluorescence excited at 305 nm (Kuramitsu et al. (1978) J. Biochem. 83, 159--170). The difference spectra of human lysozyme in the alkaline pH region were characteristic of tyrosyl ionization. The perturbation of tryptophyl residues, which had been observed for hen and turkey lysozymes (Kuramitsu & Hamaguchi (1979) J. Biochem. 85, 443--456), was not observed for human lysozyme. On the basis of the pH dependence curves of the extinction difference at 245 and and 295 nm, we roughly estimated the apparent pK values of the six tyrosyl residues as 9.2 9.2, 10.5, 10.9, 12.4, and 12.5. A time-dependent spectral change observed above pH 11 was not due to the exposure of buried tyrosyl residues on alkali denaturation but was due mainly to disulfide cleavage and exposure of buried tryptophyl residues.

BackgroundSudden sensorineural hearing loss (SSHL) is a common condition in which patients lose the hearing in one ear within 3 days. Systemic glucocorticoid treatments have been used as standard therapy for SSHL; however, about 20% of patients do not respond. We tested the safety and efficacy of topical insulin-like growth factor 1 (IGF1) application using gelatin hydrogels as a treatment for SSHL.MethodsPatients with SSHL that showed no recovery to systemic glucocorticoid administration were recruited. We applied gelatin hydrogels, impregnated with recombinant human IGF1, into the middle ear. The primary outcome measure was the proportion of patients showing hearing improvement 12 weeks after the test treatment. The secondary outcome measures were the proportion of patients showing improvement at 24 weeks and the incidence of adverse events. The null hypothesis was that 33% of patients would show hearing improvement, as was reported for a historical control after hyperbaric oxygen therapy.ResultsIn total, 25 patients received the test treatment at a median of 23 days (range 15-32) after the onset of SSHL, between 2007 and 2009. At 12 weeks after the test treatment, 48% (95% CI 28% to 69%; P = 0.086) of patients showed hearing improvement, and the proportion increased to 56% (95% CI 35% to 76%; P = 0.015) at 24 weeks. No serious adverse events were observed.ConclusionsTopical IGF1 application using gelatin hydrogels is well tolerated and may be efficacious for hearing recovery in patients with SSHL that is resistant to systemic glucocorticoids.

The 33-kDa protein is one of the three extrinsic proteins in the oxygen-evolving photosystem II complexes. The protein has one intrachain disulfide bond. On reduction of this disulfide bond, the protein was unfolded and lost its activity. On the basis of the unfolding equilibrium curve obtained by using guanidine hydrochloride, the free energy change of unfolding in the absence of guanidine hydrochloride was estimated to be 4.4 kcal/mol using the Tanford method [Tanford, C. (1970) Adv. Protein Chem. 24, 1-95] and 2.8 kcal/mol using the linear extrapolation method. The unfolding of the 33-kDa protein caused by reduction was explained in terms of the entropy change associated with reduction of the intrachain disulfide bond. The kinetics of the reduction of the disulfide bond using dithiothreitol were studied at various concentrations of guanidine hydrochloride at pH 7.5 and 25 degrees C. The disulfide bond was reduced even in the absence of guanidine hydrochloride. The unfolding and refolding kinetics of the 33-kDa protein using guanidine hydrochloride were also studied under the same conditions, and the results were compared with those for the reduction kinetics. It was shown that the reduction of the disulfide bond proceeds through a species in which the disulfide bond is exposed by local fluctuations.